Dendritic cell potentiation

ABSTRACT

A molecule capable of potentiating immune responses is described, as well as methods for using the molecule to enhance immune responses and enhance dendritic cell function. Also described are compositions containing the molecule and methods for using the compositions to treat or immunize individuals.

TECHNICAL FIELD

This invention relates to methods for enhancing immune responses, andmore particularly to enhancing immune responses by prolonging dendriticcell longevity.

BACKGROUND

Decavalent IgM antibodies display measurable binding avidity toantigens, even though binding affinity may be low. The multivalentstructure of pentameric IgM provides the potential for cross-linkingcell surface targets, endowing the soluble antibodies with biologicalpotential not normally associated with immune function.

Dendritic cells are efficient antigen-presenting cells (APC). Thesecells express class I and class II major histocompatibility complex(MHC) peptide-presenting molecules on their cell surfaces, along with aseries of costimulatory molecules (Banchereau and Steinman (1998) Nature392:245–252). Naïve T cells express receptors for these dendritic cellligands. Following recognition of peptide-antigen presented in thecontext of class I or class II molecules, the structure of the T cellmembrane is reorganized, bringing together the elements of the T cellreceptor with other cell-surface molecules, including the co-receptorsCD4 or CD8 and the costimulatory receptors CD28 and CTLA-4 (Monks et al.(1998) Nature 395:82–86; and Wulfing and Davis (1998) Science282:2266–2269). Interactions within the newly formed macromolecularcomplexes determine the outcome of inductive events transduced into Tcells by dendritic cells.

Dendritic cells reside in a variety of tissues and display distincttissue-associated phenotypes (Strunk et al. (1997) J. Exp. Med.185:1131–1136; Caux et al. (1996) J. Exp. Med. 184:695–706; Wu et al.(1996) J. Exp. Med. 184:903–911; and Vremec et al. (1992) J. Exp. Med.176:47–58). The relationships among the cell lineages of these differentsubsets of cells are not firmly established. A large body of work hasemerged focusing on dendritic cells generated in vitro from bone marrowor blood precursors (Mayordomo et al. (1995) Nat. Med. 1:1297–1302;Nonacs et al. (1992) J. Exp. Med. 176:519–529; Steinman and Witmer(1978) Proc. Natl. Acad. Sci. USA 75:5132–5136; and Young and Steinman(1990) J. Exp. Med. 171:1315–1332). The cells generated in vitro expresshigh levels of class I antigens and the series of costimulatory ligandsassociated with endogenous dendritic cells (Fagnoni et al. (1995)Immunology 85:467–474; and Bancereau et al. (2000) Annu. Rev. Immunol.18:767-811). Importantly, they are able to efficiently activate naïve Tcells, a function that is the signature of the dendritic cell. A methodfor clinically promoting dendritic cells to stimulate T cell activationwould be useful to treat immunocompromised patients.

SUMMARY

A challenge in harnessing the immune response is to direct immunityagainst specific antigens associated with tumors or known pathogens.Often, however, these antigens are weak and do not result in a strongimmune response. The invention described herein provides a direct way touse purified or recombinant proteins to induce potent immune responses.The procedure can be used to generate vaccines to a variety ofpathogens, and to bolster immunotherapy protocols for the treatment ofexisting diseases where the objective is to increase immuneresponsiveness.

This invention is based on the identification of a human IgM antibody(sHIgM12) that binds mouse dendritic cells and can induce dramaticimmunopotentiation. This antibody has the ability to potentiate immuneresponses against protein or tumor antigens when administered withoutother adjuvants. The antibody induces intracellular signaling changes indendritic cells grown and differentiated in vitro, and protectsdendritic cells from death caused by deprivation of growth and survivalfactors. Antibodies such as sHIgM12 and other molecules having similarfunctions therefore may have substantial therapeutic value, and inparticular can be used to enhance immune responses against pathogens andcancers. These molecules also are useful in vaccination of humans andother mammals against tumors and pathogens by potentiating the immunityof individuals to purified proteins.

The invention also is based on the discovery that the programmed death-1(PD-1) polypeptide also can enhance the function of dendritic cells.Dendritic cells incubated with immobilized PD-1, for example, can beadministered to a subject in order to potentiate an immune response.Polypeptides such as immobilized PD-1 therefore also may havesubstantial therapeutic value.

The invention features a purified molecule that binds specifically toB7-DC polypeptides on a cell. The binding can result in cross-linking ofa plurality of B7-DC polypeptides, and the molecule can potentiate animmune response upon administration to a mammal. The purified moleculecan be a polypeptide such as an antibody. The antibody can be an IgMantibody. The IgM antibody can have the epitope specificity of sHIgM12,or can be sHIgM12. The antibody can be a cross-linked, multivalent IgG,IgA, IgD, or IgE complex. The purified molecule can be a polypeptide(e.g., PD-1) immobilized on a solid substrate. The cell can be adendritic cell or a tumor cell (e.g., a glioma tumor cell). Theadministration can be by injection. The mammal can be a human.

In another aspect, the invention features a purified molecule that bindsspecifically to B7-DC polypeptides on a cell, wherein such binding canresult in cross-linking of a plurality of said B7-DC polypeptides, andwherein dendritic cells contacted with the molecule exhibit at least onecharacteristic selected from the group consisting of prolongedlongevity, increased NF-κB expression, increased NF-κB translocation tothe nucleus, increased ability to activate naïve T cells, increasedphosphorylation of Akt, increased localization to lymph nodes uponadministration to a mammal, maintenance of metabolic rate in cultureafter cytokine withdrawal, and increased IL-12 secretion. The activationof naïve T cells can be measured by detection of one or both of CD44 andCD69 on the T cells, or by incorporation of ³H-thymidine into the Tcells.

In another aspect, the invention features a composition containing thepurified molecule. The composition can further contain an antigen,wherein the antigen is capable of eliciting an immune response when thecomposition is administered to a mammal. The antigen can be a tumorantigen or an antigen from a pathogen. The antigen can be a component ofa killed virus or a component of a killed bacterium. The antigen can bea killed virus or a killed bacterium. The mammal can be a human. Thecomposition can further contain dendritic cells.

In yet another aspect, the invention features a composition containing apolypeptide immobilized on a solid substrate. The immobilizedpolypeptide can bind specifically to and cross-link a plurality of B7-DCpolypeptides on a cell. Dendritic cells contacted with the immobilizedpolypeptide can exhibit at least one characteristic selected from thegroup consisting of prolonged longevity, increased NF-κB expression,increased NF-κB translocation to the nucleus, increased ability toactivate naïve T cells, increased phosphorylation of Akt, increasedlocalization to lymph nodes upon administration to a mammal, maintenanceof metabolic rate in culture after cytokine withdrawal, and increasedIL-12 secretion. The immobilized polypeptide can be PD-1. Thecomposition can further contain dendritic cells.

The invention also features a method of enhancing dendritic cellfunction in a mammal. The method can include administering to the mammala composition of the invention. The enhancing of dendritic cell functioncan involve prolonging the longevity of the dendritic cells.

In another aspect, the invention features a method of potentiating animmune response in a mammal. The method can include administering to themammal a composition of the invention.

The invention also features a method of treating a tumor in a mammal.The method can involve administering to the mammal a composition of theinvention.

In yet another aspect, the invention features a method of inducingimmunity to a pathogen in a mammal. The method can include administeringa composition of the invention to the mammal.

In another aspect, the invention features a method of potentiating animmune response in a mammal. The method can include contacting dendriticcells of mammal in vitro with a composition of the invention, andtransferring the contacted dendritic cells into the mammal.

The invention also features an isolated nucleic acid encoding themolecule of the invention, a vector containing the nucleic acid of theinvention, and a host cell containing the vector of the invention.

In yet another aspect, the invention features articles of manufacturecontaining the molecules and compositions of the invention. The articlesof manufacture can further contain an antigen. The antigen can becapable of eliciting an immune response.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a series of fluorescence-activated cell sorting (FACS) plotsshowing the staining of bone marrow-derived murine dendritic cells withsHIgM12 or polyclonal human IgM as indicated (upper panels), followed byfluorescein isothiocyanate- (FITC-) conjugated α-human IgM. Cells alsowere costained with phycoerythrin- (PE-) conjugated α-CD11c. The lowerpanels show staining with antibodies against typical dendritic cellsurface markers, including MHC class II and CD86.

FIG. 2 shows are line graphs showing the effect of sHIgM12-treated,antigen-pulsed dendritic cells on incorporation of ³H-thymidine into Tcells, an indicator of proliferation. The lop graph shows the MHC classI-restricted T cell response to dendritic cells treated with sHIgM12(filled circles) or control polyclonal human IgM control (HIgM; opencircles). The bottom graph shows the MHC class II-restricted T cellresponse to dendritic cells treated with sHIgM12 (filled circles) orcontrol polyclonal HIgM (open circles).

FIG. 3 is a line graph showing the effect of sHIgM12 treated,antigen-pulsed dendritic cells on in vivo T cell priming. Mousedendritic cells were pulsed with ovalbumin and treated with sHIgM12(open circles) or polyclonal HIgM control (closed circles) prior toadoptive transfer. The data depict levels of 3H incorporation intosplenocytes that were harvested 7 days after adoptive transfer andtreated with titrating doses of ovalbumin.

FIG. 4A is a histogram showing amounts of dendritic cell staining bysHIgM12 (gray filled histogram), polyclonal HIgM control (black filledhistogram), and monomeric sHIgM12 (thick black line outlining unfilledhistogram). FIG. 4B is a line graph showing levels of activation ofnaïve OT-1 splenocytes by dendritic cells treated with pentamericsHIgM12 (filled circles), polyclonal HIgM control (open circles),monomeric sHIgM12 (open squares), and monomeric sHIgM12 followed bypentameric sHIgM12 (open triangles).

FIG. 5A is a histogram showing levels of sHIgM12 staining of dendriticcells (gray histogram) or dendritic cells preincubated with PK-1.Ig(unshaded histogram). A control antibody (black histogram) did not stainthe cells. FIG. 5B is a histogram of the reciprocal experiment, showingPD-1.Ig staining of dendritic cells (gray histogram) or dendritic cellspreincubated with sHIgM12 (unshaded histogram).

FIG. 6 is a line graph showing the immune response of splenocytes frommice that were treated in vivo with sHIgM12 (filled circles) orpolyclonal HIgM control (open circles) before being isolated andimmunized with ovalbumin.

FIG. 7A is a column graph showing levels of dendritic cell metabolismbefore and after cytokine withdrawal from untreated cells and cellstreated with either sHIgM12 or a control antibody as indicated. FIG. 7Bis a column graph showing levels of dendritic cell metabolism before andafter cytokine withdrawal from untreated cells and cells treated witheither immobilized PD-1.Ig, sHIgM12, or a control antibody as indicated.

FIG. 8 is a map of an expression vector that can be used to produceantibodies.

DETAILED DESCRIPTION

1. Molecules

The invention provides molecules that bind specifically to B7-DCpolypeptides. Such molecules can bind simultaneously to a plurality ofB7-DC polypeptides (i.e., one such molecule can bind to more than oneB7-DC polypeptide at the same time). Molecules provided herein thus caneffectively cross-link a plurality of B7-DC polypeptides. Molecules ofthe invention typically are polypeptides, and antibodies can beparticularly useful (see below).

Molecules of the invention can bind specifically to cells through B7-DCpolypeptides that are present on the cell surface. As used herein,“binds specifically to B7-DC” means that a molecule binds preferentiallyto B7-DC and does not display significant binding to other cell surfacepolypeptides (e.g., substantially less, or no, detectable binding toother cell surface polypeptides). Molecules (e.g., antibodies orpolypeptides) can be tested for recognition of B7-DC by standardimmunoassay methods, including FACS, enzyme-linked immunosorbent assay(ELISA), and radioimmuno assay (RIA). See, e.g., Short Protocols inMolecular Biology, eds. Ausubel et al., Green Publishing Associates andJohn Wiley & Sons (1992).

B7-DC is a cell surface polypeptide that can be found on, for example,dendritic cells and some tumor cells (e.g., glioma tumor cells).Molecules of the invention can bind to B7-DC on the surface of dendriticcells in a mammal (e.g., a human) and potentiate an immune response. Asused herein, the term “potentiate an immune response” encompassesenhancement of dendritic cell function and increased activation of naïveT cells. Enhanced dendritic cell function includes components such asprolonged longevity of dendritic cells, which can be detected based onincreased expression of NF-κB and increased translocation of NF-κB tothe nucleus. Other components of enhanced dendritic cell functioninclude an increased ability of dendritic cells to activate naïve Tcells, increased localization of dendritic cells to the lymph nodes,increased phosphorylation of Akt (also known as protein kinase B) withindendritic cells, and increased secretion of interleukin-12 (IL-12) bydendritic cells. Molecules provided by the invention also can enhancethe metabolism of dendritic cells upon the withdrawal of cytokines fromdendritic cells in culture. The molecules described herein can beadministered to a mammal (e.g., a human) in order to enhance dendriticcell function and potentiate an immune response that can include any orall of the above-listed components. Molecules of the invention also canbe used to contact and activate dendritic cells in vitro.

Potentiation of an immune response by molecules of the invention can bemeasured by assessing any of the components listed above. Secretion ofIL-12 can be measured, for example, by an enzyme linked immunosorbent(ELISA) assay as described in the Examples (below). Activation of naïveT cells can be assayed by, for example, measuring the incorporation of³H-thymidine into newly synthesized DNA in proliferating cells, or bydetecting T cell activation markers such as CD44 and/or CD69. Expressionor translocation of NF-κB can be measured by, for example, cell stainingwith an antibody against NF-κB. Increased phosphorylation of Akt can beassessed by, for example, western blotting with an antibody againstphosphorylated Akt. Antibodies against NF-κB and phosphorylated Akt areavailable from, for example, Cell Signaling Technologies, Inc. (Beverly,Mass.). Methods for measuring the other components encompassed byenhanced dendritic cell function and immunopotentiation also aredescribed herein.

The molecules provided by the invention typically are purified. The term“purified” as used herein refers to a molecule that has been separatedor isolated from other cellular components by which it is naturallyaccompanied (e.g., other cellular proteins, polynucleotides, or cellularcomponents), or separated from other components present in a reactionmixture when the molecule is synthesized in vitro. “Purified” as usedherein also encompasses molecules that are partially purified, so thatat least some of the components by which the molecule is accompanied areremoved. Typically, a molecule is considered “purified” when it is atleast 50% (e.g., 55%, 60%, 70%, 80%, 90%, 95%, or 99%), by dry weight,free from the proteins and other organic molecules or components withwhich it naturally associates or with which it is accompanied in asynthesis reaction.

2. Polypeptides and Antibodies

Molecules of the invention can be polypeptides. As used herein, apolypeptide is an amino acid chain, regardless of length orpost-translational modification (e.g., phosphorylation orglycosylation). The polypeptides provided herein can bind specificallyto B7-DC, and upon administration to a mammal (e.g., a human), canenhance dendritic cell function and potentiate an immune response.Polypeptides of the invention also can enhance dendritic cell functionwhen incubated in vitro with dendritic cells.

PD-1 is a polypeptide that is a natural receptor for B7-DC. PD-1 can beimmobilized on a solid substrate (e.g., a plastic dish or a glassmicroscope slide). Upon incubation with dendritic cells, immobilizedPD-1 can cross-link a plurality of B7-DC polypeptides on the cellsurface and enhance the function of the dendritic cells. Incubation ofcultured dendritic cells with immobilized PD-1 can, for example,maintain the metabolic rate of the cells upon removal of cytokines fromthe culture medium, as compared to the metabolic rate of dendritic cellsthat are not incubated with PD-1 (see Example 7).

Molecules of the invention can be antibodies that have specific bindingactivity for B7-DC. The terms “antibody” and “antibodies” encompassintact molecules as well as fragments thereof that are capable ofbinding to B7-DC. An antibody can be of any immunoglobulin (Ig) class,including IgM, IgA, IgD, IgE, and IgG, and any subclass thereof.Antibodies of the IgM class (e.g., sHIgM12) typically are pentavalentand are particularly useful because one antibody molecule can cross-linka plurality of B7-DC polypeptides. Immune complexes containing Igmolecules that are cross-linked (e.g., cross-linked IgG) and are thusmultivalent also are capable of cross-linking a plurality of B7-DCmolecules, and can be particularly useful.

As used herein, an “epitope” is a portion of an antigenic molecule towhich an antibody binds. Antigens can present more than one epitope atthe same time. For polypeptide antigens, an epitope typically is aboutfour to six amino acids in length. Two different immunoglobulins canhave the same epitope specificity if they bind to the same epitope orset of epitopes.

The terms “antibody” and “antibodies” include polyclonal antibodies,monoclonal antibodies, humanized or chimeric antibodies, single chain Fvantibody fragments, Fab fragments, and F(ab)₂ fragments. Polyclonalantibodies are heterogeneous populations of antibody molecules that arespecific for a particular antigen, while monoclonal antibodies arehomogeneous populations of antibodies to a particular epitope containedwithin an antigen.

Polyclonal antibodies are contained in the sera of immunized animals.Monoclonal antibodies can be prepared using, for example, standardhybridoma technology. In particular, monoclonal antibodies can beobtained by any technique that provides for the production of antibodymolecules by continuous cell lines in culture as described, for example,by Kohler et al. (1975) Nature 256:495–497, the human B-cell hybridomatechnique of Kosbor et al. (1983) Immunology Today 4:72, and Cote et al.(1983) Proc. Natl. Acad. Sci. USA 80:2026–2030, and the EBV-hybridomatechnique of Cole et al., Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc. pp. 77–96 (1983). A hybridoma producing monoclonalantibodies of the invention can be cultivated in vitro or in vivo.

Antibodies of the invention also can be isolated from, for example, theserum of an individual. The sHIgM12 antibody, for example, was isolatedfrom human serum as described in Example 1 herein. Suitable methods forisolation include purification from mammalian serum using techniquesthat include, for example, chromatography.

Antibodies that bind to B7-DC also can be produced by, for example,immunizing host animals (e.g., rabbits, chickens, mice, guinea pigs, orrats) with B7-DC. A B7-DC polypeptide or a portion of a B7-DCpolypeptide can be produced recombinantly, by chemical synthesis, or bypurification of the native protein, and then used to immunize animals byinjection of the polypeptide. Adjuvants can be used to increase theimmunological response, depending on the host species. Suitableadjuvants include Freund's adjuvant (complete and incomplete), mineralgels such as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanin (KLH), and dinitrophenol. Standard techniquescan be used to isolate antibodies generated in response to the B7-DCimmunogen from the sera of the host animals. Such techniques are usefulfor generating antibodies that have similar characteristics to sHIgM12(e.g., similar epitope specificity and other functional similarities).

Antibodies such as sHIgM12 also can be produced recombinantly. The aminoacid sequence (e.g., the partial amino acid sequence) of an antibodyprovided herein can be determined by standard techniques, and a cDNAencoding the antibody or a portion of the antibody can be isolated fromthe serum of the subject (e.g., the human patient or the immunized hostanimal) from which the antibody was originally isolated. The CDNA can becloned into an expression vector using standard techniques. Theexpression vector then can be transfected into an appropriate host cell(e.g., a Chinese hamster ovary cell, a COS cell, or a hybridoma cell),and the antibody can be expressed and purified. See, for example,Example 9 herein.

Antibody fragments that have specific binding affinity for B7-DC andretain cross-linking function also can be generated by techniques suchas those disclosed above. Such antibody fragments include, but are notlimited to, F(ab′)₂ fragments that can be produced by pepsin digestionof an antibody molecule, and Fab fragments that can be generated byreducing the disulfide bridges of F(ab′)₂ fragments. Alternatively, Fabexpression libraries can be constructed. See, for example, Huse et al.(1989) Science 246:1275–1281. Single chain Fv antibody fragments areformed by linking the heavy and light chain fragments of the Fv regionvia an amino acid bridge (e.g., 15 to 18 amino acids), resulting in asingle chain polypeptide. Single chain Fv antibody fragments can beproduced through standard techniques, such as those disclosed in U.S.Pat. No. 4,946,778. Such fragments can be rendered multivalent by, forexample, biotinylation and cross-linking, thus generating antibodyfragments that can cross-link a plurality of B7-DC polypeptides.

3. Nucleic Acids, Vectors, and Host Cells

The invention provides nucleic acids encoding molecules (e.g.,polypeptides and antibodies) that bind specifically to B7-DC. As usedherein, the term “nucleic acid” refers to both RNA and DNA, includingcDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. Anucleic acid molecule can be double-stranded or single-stranded (i.e., asense or an antisense single strand). Nucleic acids of the inventioninclude, for example, cDNAs encoding the light and heavy chains of thesHIgM12 antibody.

An “isolated nucleic acid” refers to a nucleic acid that is separatedfrom other nucleic acid molecules that are present in a vertebrategenome, including nucleic acids that normally flank one or both sides ofthe nucleic acid in a vertebrate genome. The term “isolated” as usedherein with respect to nucleic acids also includes anynon-naturally-occurring nucleic acid sequence, since suchnon-naturally-occurring sequences are not found in nature and do nothave immediately contiguous sequences in a naturally-occurring genome.

An isolated nucleic acid can be, for example, a DNA molecule, providedone of the nucleic acid sequences normally found immediately flankingthat DNA molecule in a naturally-occurring genome is removed or absent.Thus, an isolated nucleic acid includes, without limitation, a DNAmolecule that exists as a separate molecule (e.g., a chemicallysynthesized nucleic acid, or a cDNA or genomic DNA fragment produced byPCR or restriction endonuclease treatment) independent of othersequences as well as DNA that is incorporated into a vector, anautonomously replicating plasmid, a virus (e.g., a retrovirus,lentivirus, adenovirus, or herpes virus), or into the genomic DNA of aprokaryote or eukaryote. In addition, an isolated nucleic acid caninclude an engineered nucleic acid such as a DNA molecule that is partof a hybrid or fusion nucleic acid. A nucleic acid existing amonghundreds to millions of other nucleic acids within, for example, cDNAlibraries or genomic libraries, or gel slices containing a genomic DNArestriction digest, is not considered an isolated nucleic acid.

The isolated nucleic acid molecules provided herein can be produced bystandard techniques, including, without limitation, common molecularcloning and chemical nucleic acid synthesis techniques. For example,polymerase chain reaction (PCR) techniques can be used to obtain anisolated nucleic acid molecule encoding sHIgM12. Isolated nucleic acidsof the invention also can be chemically synthesized, either as a singlenucleic acid molecule (e.g., using automated DNA synthesis in the 3′ to5′ direction using phosphoramidite technology) or as a series ofpolynucleotides. For example, one or more pairs of long polynucleotides(e.g., >100 nucleotides) can be synthesized that contain the desiredsequence, with each pair containing a short segment of complementarity(e.g., about 15 nucleotides) such that a duplex is formed when thepolynucleotide pair is annealed. DNA polymerase is used to extend thepolynucleotides, resulting in a single, double-stranded nucleic acidmolecule per polynucleotide pair.

The invention also provides vectors containing nucleic acids such asthose described above. As used herein, a “vector” is a replicon, such asa plasmid, phage, or cosmid, into which another DNA segment may beinserted so as to bring about the replication of the inserted segment.The vectors of the invention can be expression vectors. An “expressionvector” is a vector that includes one or more expression controlsequences, and an “expression control sequence” is a DNA sequence thatcontrols and regulates the transcription and/or translation of anotherDNA sequence.

In the expression vectors of the invention, a nucleic acid (e.g., anucleic acid encoding the light and/or heavy chains of sHIgM12) isoperably linked to one or more expression control sequences. As usedherein, “operably linked” means incorporated into a genetic construct sothat expression control sequences effectively control expression of acoding sequence of interest. Examples of expression control sequencesinclude promoters, enhancers, and transcription terminating regions. Apromoter is an expression control sequence composed of a region of a DNAmolecule, typically within 100 nucleotides upstream of the point atwhich transcription starts (generally near the initiation site for RNApolymerase II). To bring a coding sequence under the control of apromoter, it is necessary to position the translation initiation site ofthe translational reading frame of the polypeptide between one and aboutfifty nucleotides downstream of the promoter. Enhancers provideexpression specificity in terms of time, location, and level. Unlikepromoters, enhancers can function when located at various distances fromthe transcription site. An enhancer also can be located downstream fromthe transcription initiation site. A coding sequence is “operablylinked” and “under the control” of expression control sequences in acell when RNA polymerase is able to transcribe the coding sequence intomRNA, which then can be translated into the protein encoded by thecoding sequence. Expression vectors provided herein thus are useful toproduce sHIgM12, as well as other molecules of the invention.

Suitable expression vectors include, without limitation, plasmids andviral vectors derived from, for example, bacteriophage, baculoviruses,tobacco mosaic virus, herpes viruses, cytomegalovirus, retroviruses,vaccinia viruses, adenoviruses, and adeno-associated viruses. Numerousvectors and expression systems are commercially available from suchcorporations as Novagen (Madison, Wis.), Clontech (Palo Alto, Calif.),Stratagene (La Jolla, Calif.), and Invitrogen/Life Technologies(Carlsbad, Calif.).

An expression vector can include a tag sequence designed to facilitatesubsequent manipulation of the expressed nucleic acid sequence (e.g.,purification or localization). Tag sequences, such as green fluorescentprotein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc,hemagglutinin, or Flag™ tag (Kodak, New Haven, Conn.) sequencestypically are expressed as a fusion with the encoded polypeptide. Suchtags can be inserted anywhere within the polypeptide including at eitherthe carboxyl or amino terminus.

The invention also provides host cells containing vectors of theinvention. The term “host cell” is intended to include prokaryotic andeukaryotic cells into which a recombinant expression vector can beintroduced. As used herein, “transformed” and “transfected” encompassthe introduction of a nucleic acid molecule (e.g., a vector) into a cellby one of a number of techniques. Although not limited to a particulartechnique, a number of these techniques are well established within theart. Prokaryotic cells can be transformed with nucleic acids by, forexample, electroporation or calcium chloride mediated transformation.Nucleic acids can be transfected into mammalian cells by techniquesincluding, for example, calcium phosphate co-precipitation,DEAE-dextran-mediated transfection, lipofection, electroporation, ormicroinjection. Suitable methods for transforming and transfecting hostcells are found in Sambrook et al., Molecular Cloning: A LaboratoryManual (2^(nd) edition), Cold Spring Harbor Laboratory, New York (1989),and reagents for transformation and/or transfection are commerciallyavailable (e.g., Lipofectin® (Invitrogen/Life Technologies); Fugene(Roche, Indianapolis, Ind.); and SuperFect (Qiagen, Valencia, Calif.)).

4. Compositions

The invention provides compositions containing the molecules describedherein (e.g., antibodies such as sHIgM12 and polypeptides such as PD-1).Such compositions are suitable for administration to a subject toenhance dendritic cell function and potentiate an immune response. Asdescribed above, enhanced dendritic cell function includes suchcomponents as prolonged longevity, increased ability to activate naïve Tcells, increased localization to the lymph nodes, increasedphosphorylation of Akt, and increased secretion of interleukin-12(IL-12).

Compositions provided herein also can contain a molecule (e.g., PD-1)that is immobilized on a solid substrate. Such compositions can be usedto contact dendritic cells and enhance their function as describedabove.

Methods for formulating and subsequently administering therapeuticcompositions are well known to those skilled in the art. Dosagestypically are dependent on the responsiveness of the subject to themolecule, with the course of treatment lasting from several days toseveral months, or until a suitable immune response is achieved. Personsof ordinary skill in the art routinely determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages can vary dependingon the relative potency of an antibody, and generally can be estimatedbased on the EC₅₀ found to be effective in in vitro and/or in vivoanimal models. Dosage typically is from 0.01 μg to 100 g per kg of bodyweight (e.g., from 1 μg to 100 mg, from 10 μg to 10 mg, or from 50 μg to500 μg per kg of body weight). Compositions containing the moleculesprovided herein may be given once or more daily, weekly, monthly, oreven less often.

In addition to the molecules provided herein, compositions of theinvention further can contain antigens that will elicit a specificimmune response. Suitable antigens include, for example, polypeptides orfragments of polypeptides expressed by tumors and pathogenic organisms.Killed viruses and bacteria, in addition to components of killed virusesand bacteria, also are useful antigens. Such antigens can stimulateimmune responses against tumors or pathogens.

Compositions provided herein also can include dendritic cells that havebeen isolated from, for example, bone marrow, spleen, or thymus tissue.Dendritic cell lines also can be useful in compositions of theinvention.

Molecules of the invention can be admixed, encapsulated, conjugated orotherwise associated with other molecules, molecular structures, ormixtures of compounds such as, for example, liposomes, receptor targetedmolecules, or oral, topical or other formulations for assisting inuptake, distribution and/or absorption.

Pharmaceutically acceptable carriers are pharmaceutically acceptablesolvents, suspending agents, or any other pharmacologically inertvehicles for delivering antibodies to a subject. Pharmaceuticallyacceptable carriers can be liquid or solid, and can be selected with theplanned manner of administration in mind so as to provide for thedesired bulk, consistency, and other pertinent transport and chemicalproperties, when combined with one or more therapeutic compounds and anyother components of a given pharmaceutical composition. Typicalpharmaceutically acceptable carriers include, without limitation: water;saline solution; binding agents (e.g., polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars,gelatin, or calcium sulfate); lubricants (e.g., starch, polyethyleneglycol, or sodium acetate); disintegrates (e.g., starch or sodium starchglycolate); and wetting agents (e.g., sodium lauryl sulfate).

Pharmaceutical compositions containing molecules provided herein can beadministered by a number of methods, depending upon whether local orsystemic treatment is desired. Administration can be, for example,parenteral (e.g., by subcutaneous, intrathecal, intraventricular,intramuscular, or intraperitoneal injection, or by intravenous drip);oral; topical (e.g., transdermal, sublingual, ophthalmic, orintranasal); or pulmonary (e.g., by inhalation or insufflation ofpowders or aerosols). Administration can be rapid (e.g., by injection)or can occur over a period of time (e.g., by slow infusion oradministration of slow release formulations). For administration to thecentral nervous system, antibodies can be injected or infused into thecerebrospinal fluid, typically with one or more agents capable ofpromoting penetration across the blood-brain barrier.

Compositions and formulations for parenteral, intrathecal orintraventricular administration include sterile aqueous solutions (e.g.,sterile physiological saline), which also can contain buffers, diluentsand other suitable additives (e.g., penetration enhancers, carriercompounds and other pharmaceutically acceptable carriers).

Compositions and formulations for oral administration include, forexample, powders or granules, suspensions or solutions in water ornon-aqueous media, capsules, sachets, or tablets. Such compositions alsocan incorporate thickeners, flavoring agents, diluents, emulsifiers,dispersing aids, or binders.

Formulations for topical administration include, for example, sterileand non-sterile aqueous solutions, non-aqueous solutions in commonsolvents such as alcohols, or solutions in liquid or solid oil bases.Such solutions also can contain buffers, diluents and other suitableadditives. Pharmaceutical compositions and formulations for topicaladministration can include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids, and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be useful.

Pharmaceutical compositions include, but are not limited to, solutions,emulsions, aqueous suspensions, and liposome-containing formulations.These compositions can be generated from a variety of components thatinclude, for example, preformed liquids, self-emulsifying solids andself-emulsifying semisolids. Emulsion formulations are particularlyuseful for oral delivery of therapeutic compositions due to their easeof formulation and efficacy of solubilization, absorption, andbioavailability. Liposomes can be particularly useful due to theirspecificity and the duration of action they offer from the standpoint ofdrug delivery.

Molecules of the invention can encompass any pharmaceutically acceptablesalts, esters, or salts of such esters, or any other compound which,upon administration to a subject, is capable of providing (directly orindirectly) the biologically active metabolite or residue thereof.Accordingly, for example, the invention provides pharmaceuticallyacceptable salts of molecules such as antibodies (e.g., sHIgM12),prodrugs and pharmaceutically acceptable salts of such prodrugs, andother bioequivalents. A prodrug is a therapeutic agent that is preparedin an inactive form and is converted to an active form (i.e., drug)within the body or cells thereof by the action of endogenous enzymes orother chemicals and/or conditions. The term “pharmaceutically acceptablesalts” refers to physiologically and pharmaceutically acceptable saltsof the antibodies useful in methods of the invention (i.e., salts thatretain the desired biological activity of the parent antibodies withoutimparting undesired toxicological effects). Examples of pharmaceuticallyacceptable salts include, but are not limited to, salts formed withcations (e.g., sodium, potassium, calcium, or polyamines such asspermine); acid addition salts formed with inorganic acids (e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, ornitric acid); salts formed with organic acids (e.g., acetic acid, citricacid, oxalic acid, palmitic acid, or fumaric acid); and salts formedwith elemental anions (e.g., bromine, iodine, or chlorine).

Compositions additionally can contain other adjunct componentsconventionally found in pharmaceutical compositions. Thus, thecompositions also can include compatible, pharmaceutically activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or additional materials usefulin physically formulating various dosage forms of the compositions ofthe present invention, such as dyes, flavoring agents, preservatives,antioxidants, opacifiers, thickening agents, and stabilizers.Furthermore, the composition can be mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavorings,penetration enhancers, and aromatic substances. When added, however,such materials should not unduly interfere with the biologicalactivities of the PNA components within the compositions of the presentinvention.

Pharmaceutical formulations of the present invention, which can bepresented conveniently in unit dosage form, can be prepared according toconventional techniques well known in the pharmaceutical industry. Suchtechniques include the step of bringing into association the activeingredients (i.e., the antibodies) with the desired pharmaceuticalcarrier(s). Typically, the formulations can be prepared by uniformly andintimately bringing the active ingredients into association with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product. Formulations can be sterilized ifdesired, provided that the method of sterilization does not interferewith the effectiveness of the antibody(s) contained in the formulation.

Compositions of the present invention can be formulated into any of manypossible dosage forms such as, without limitation, tablets, capsules,liquid syrups, soft gels, suppositories, and enemas. Compositions alsocan be formulated as suspensions in aqueous, non-aqueous or mixed media.Aqueous suspensions further can contain substances that increase theviscosity of the suspension including, for example, sodiumcarboxymethyl-cellulose, sorbitol, and/or dextran. Suspensions also cancontain stabilizers.

5. Methods of Using the Molecules Provided Herein to Potentiate anImmune Response

The invention provides methods for using molecules described herein toenhance dendritic cell function and potentiate an immune response.Molecules of the invention can interact specifically with B7-DC and, asdescribed herein, can enhance the function of dendritic cells andpotentiate an immune response. Methods of the invention are particularlyuseful for treating tumors and inducing immunity to a specific antigen.

The methods provided herein typically involve administering to a mammal(e.g., a human) a molecule of the invention (e.g., an antibody such assHIgM12) or a composition of the invention (e.g., a compositioncontaining sHIgM12). Methods of the invention also involveadministration of dendritic cells that have been contacted with amolecule or a composition provided herein (e.g., a compositionscontaining sHIgM12 and an antigen). Such dendritic cells are useful topotentiate an immune response in the mammal to which they areadministered.

As described above, the molecule, composition, or activated dendriticcells can be administered by any suitable systemic or local method.Systemic methods of administration include, without limitation, oral,topical, or parenteral administration, as well as administration byinjection. Local methods of administration include, for example, directinjection into a tumor.

Methods of the invention can be used to enhance dendritic cell function.The enhancement of dendritic cell function includes, for example,prolonging the longevity of dendritic cells, increasing the ability ofdendritic cells to activate naïve T cells, and increasing thelocalization of dendritic cells to lymph nodes in a mammal. Thelongevity of dendritic cells can be assessed by, for example, measuringthe expression of NF-κB or the translocation of NF-κB to the nucleus.Since NF-κB is an intracellular signal involved in the inhibition ofprogrammed cell death, increased expression or translocation of NF-κBindicates inhibition of apoptosis and prolonged dendritic celllongevity. T cell activation can be measured by, for example, assessingthe incorporation of radiolabeled (e.g., tritiated) thymidine into newlysynthesized DNA in proliferating T cells. Activation of naïve T cellsalso can be measured by detecting (e.g., by flow cytometry) CD44 and/orCD69 activation markers on the T cell surface.

Methods for potentiating an immune response (i.e., inducing immunity toa particular antigen) can involve administering to a mammal (e.g., ahuman) a composition that contains (1) a purified molecule (e.g., apolypeptide or an antibody, particularly sHIgM12) capable of bindingspecifically to B7-DC polypeptides, and (2) an antigen (e.g., an antigenfrom a tumor cell or from a pathogen). Such methods also can involveadministering dendritic cells that have been activated in vitro bycontacting the cells with (1) a purified molecule (e.g., a polypeptideor an antibody such as sHIgM12) capable of binding specifically to B7-DCpolypeptides, and (2) an antigen (e.g., an antigen from a tumor cell orfrom a pathogen). These methods are useful to, for example, treat tumorsand/or induce immunity to pathogens.

Methods of the invention can be useful for treating solid tumorsincluding, without limitation, breast cancer, lung cancer, pancreaticcancer, brain cancer, prostate cancer, ovarian cancer, uterine cancer,renal cancer, melanoma, and other solid tumors. Such methods areparticularly useful for treating melanoma and renal carcinoma tumors. Asolid tumor can be, for example, an early-stage solid tumor. As usedherein, the term “treating a tumor” encompasses reducing the size of atumor, reducing the number of viable cells in a tumor, and/or slowing orstopping the growth of a tumor. Methods for assessing such outcomes areknown in the art. Methods for treating tumors can involve administrationof a molecule or composition of the invention (e.g., a compositioncontaining sHIgM12 and a tumor antigen) either systemically (e.g.,intravenously or subcutaneously) or directly to a tumor (e.g., byinjection).

7. Articles of Manufacture

The invention provides articles of manufacture that can include themolecules and/or compositions provided herein. The molecules and/orcompositions can be combined with packaging material and sold as kitsfor potentiating an immune response in an individual. Components andmethods for producing articles of manufacture are well known. Articlesof manufacture may combine one or more of the molecules set out in theabove sections. An article of manufacture can contain a composition thatincludes a molecule provided herein (e.g., an antibody such as sHIgM12or a polypeptide such as immobilized PD-1). An article of manufacturealso can include one or more antigens (e.g., a tumor antigen or anantigen from a pathogen) that can stimulate a specific immune response.Furthermore, an article of manufacture can contain dendritic cells. Anarticle of manufacture also may include, for example, buffers or othercontrol reagents for potentiating an immune response. Instructionsdescribing how the molecules, antigens, dendritic cells, andcompositions are effective for potentiating an immune response can beincluded in such kits.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Materials and Methods

Isolation of human antibodies—Human serum samples were obtained from thedysproteinemia clinic, and those exhibiting an Ig clonal peak of greaterthan 20 mg/ml were chosen for further evaluation. The selected sampleswere from 50 patients with a wide variety of conditions characterized bya monoclonal IgM spike, including Waldenstrom's macroglobulinemia,lymphoma, and monoclonal gammopathy of undetermined significance. Serawere dialyzed against water, and precipitates were collected bycentrifugation at 14,000 rpm for 30 minutes and dissolved in phosphatebuffered saline (PBS). The samples were centrifuged and chromatographedon a Superose-6 column (Amersham Pharmacia, Piscataway, N.J.). IgMfractions were pooled and analyzed by SDS-PAGE, and proteinconcentrations were determined by reading absorbance at 280 nm. IgMsolutions were sterile filtered and cryopreserved. The antibody sHIgM12was identified based on its ability to bind dendritic cells asdetermined by FACS analysis (see Example 2). The polyclonal human IgMantibody control was described previously (Miller et al. (11994) J.Neurosci. 14:6230–6238).

Monomeric sHIgM12 was obtained from the pentameric form by reductionwith 5 mM dithiothreitol (Sigma-Aldrich, St. Louis, Mo.) in 200 mM Tris,150 mM NaCl, 1 mM EDTA pH 8.0 for 2 hours at room temperature.Subsequent alkylation was performed with 12 mM iodacetamide for 1 houron ice. IgM monomers were isolated by chromatography on a Superdex-200column (Amersham Pharmacia) equilibrated with PBS, and characterized byreducing and non-reducing SDS-PAGE.

Mice and reagents–C57BL6/J, C3H/HeJ, and BALB/C mouse strains wereobtained from The Jackson Laboratory (Bar Harbor, Me.). OT-1 and D0-11transgenic mouse strains (Hogquist et al. (1994) Cell 76:17–27; andMurphy et al. (1990) Science 250:1720–1723) were bred and maintained atthe Mayo Clinic animal facility according to the protocol approved bythe Institutional Review Board for Animal Rights, Mayo Clinic.C57BL/6-RAG^(−/−) mice, CD4^(−/−) mice, and GFP transgenic mice werepurchased from The Jackson Laboratory (Bar Harbor, Me.). K⁻D⁻ mice wereobtained from Francois Lemmonier, Pasteur Institute, Paris. Chickenovalbumin was obtained from Sigma-Aldrich. Peptides were synthesized atthe Mayo Protein Core Facility. Fluorophore-coupled anti-CD11c(HL-3),anti-B220(RA3-6B2), anti-CD80(16-10A1), anti-CD86(GL-1), anti-CD44(IM7),anti-CD 69(H1.2F3), anti-CD3e(145-2C11), anti-Mac1(M1/70), Pan-NKantibody(DX-5), anti-K^(b)(AF6-88.5), and anti-I-A^(b)(KH74) wereobtained from BD PharMingen (San Diego, Calif.). FITC-coupled goatanti-human IgM was obtained from Jackson ImmunoResearch Laboratories,Inc. (West Grove, Pa.). The K^(b)-Ser-Ile-Ile-Asn-Phe-Glu-Lys-Leu (SEQID NO:1) tetramer coupled to APC was prepared as previously described(Block et al. (2001) J. Immunol. 167:821–826). RPMI-1640 medium waspurchased from Gibco/Invitrogen (Carlsbad, Calif.).

Generation of immature and mature dendritic cells in vivo—Dendriticcells from bone marrow were isolated using an established protocol.Briefly, bone marrow was isolated from mouse hind leg long bones.Erythrocytes were lysed by treatment with ammonium chloride-potassiumchloride (ACK; 0.1 M NH₄Ac, 0.01 M KHCO₃, 60 μM EDTA) at 37° C. Theremaining cells were plated at a density of 1×10⁶ cells per ml in 6 wellplates (Becton Dickinson, Franklin Lakes, N.J.) in RPMI-10 containing 10μg/ml murine granulocyte macrophage-colony stimulating factor (GM-CSF;PeproTech, Inc., Rocky Hill, N.J.) and 1 ng/ml murine interleukin-4(IL-4; PeproTech, Inc.). Cells were incubated at 37° C. with 5% CO₂. Onculture day 2, cells were gently washed and the media was replaced withfresh RPMI-10 containing the same concentrations of GM-CSF and IL-4, andthe culture was continued for another 5 days. Dendritic cells werematured by the addition of either 10 μg/ml lipopolysaccharide (LPS;Difco®) or 50 μmol/ml CpG (Mayo Molecular Core Facility) to the culturesfor 48 hours. Maturation status was confirmed by staining for Class-11,CD80, and CD86.

Human dendritic cells were derived from monocytes cultured with GM-CSFand IL-4. Day 7 cells were activated with either LPS (10 μg/ml), tumornecrosis factor-α/interleukin-1β (TNF-α/IL-1β; 1.1×10⁴ U/ml and 3.2×10³U/ml, respectively), interferon-γ (IFN-γ; 2×10³ U/ml), or PBS controlfor 3 days. Dendritic cell development was followed by monitoring thepresence of the CD83 cell surface marker, as described below.

Flow cytometry—Cells were washed with fluorescence activated cellsorting (FACS) buffer (0.5% bovine serum albumin (BSA) and 0.1% sodiumazide in PBS) and centrifuged into a 96-well plate (Nunc). Antibodieswere added to the wells for a 30 minute incubation on ice. After threewashes with FACS buffer, cells were fixed with 1% paraformaldehyde andanalyzed on a FACS Calibur (Becton Dickinson). Data were analyzed usingCell Quest software (BD PharMingen).

Activated human dendritic cells were stained with 10 μg/ml of sHIgM12 orpolyclonal hIgM control on culture day 10 (3 days after induction ofmaturation). FITC-conjugated anti-hIgM secondary antibody was addedafter several washes. CD83 is a maturation marker on dendritic cells,and was assessed by anti-human CD83-PE antibody.

Human TP365 glioma cells were obtained from Dr. Robert Jenkins at theMayo Clinic (Rochester, Minn.). Cells were stained with 10 μg/ml sHIgM12or polyclonal hIgM control. A secondary anti-human IgM, Fc_(5μ),fragment specific FITC-conjugated antibody (Jackson ImmunoresearchLaboratories) was added after 2 washes. Cells subsequently were washedand fixed with 2% paraformaldehyde, and subjected to flow cytometryanalysis.

Isolation of endogenous dendritic cells—Dendritic cells were isolatedfrom mouse spleen and thymus. Tissues were cut into small pieces andincubated in RPMI containing 2 mg/ml collagenase (Sigma-Aldrich), 100μg/ml DNAse (Sigma-Aldrich), and 2% fetal calf serum (Hyclone) for 20minutes at 37° C. EDTA (0.031 M) was added for 5 minutes. Erythrocyteswere lysed with ACK at 37° C., and the remaining cells were counted andused for flow cytometry.

In vitro activation of naïve T cells—Naïve splenocytes were harvestedfrom mice and plated in triplicate after erythrocyte lysis using ACKbuffer. 3×10⁵ responder cells were stimulated in vitro for three dayswith titrating doses of antigen or antigen-pulsed dendritic cells. Theplated cells were pulsed with ³H-thymidine during the final 18 hoursbefore they were harvested and ³H levels determined.

Adoptive transfer of dendritic cells and T cells—Dendritic cells derivedfrom seven-day bone marrow cultures were pulsed overnight with 1 μmol/mlof the class-I restricted peptide Ser-Ile-Ile-Asn-Phe-Glu-Lys-Leu (SEQID NO:1) or the class-II restricted peptideIle-Ser-Gln-Ala-Val-His-Ala-Ala-His-Ala-Glu-Ile-Asn-Glu (SEQ ID NO:2),or with 1 mg/ml chicken ovalbumin. The control antibody or sHIgM12 wasco-incubated with the peptide in the cultures at a concentration of 10μg/ml. Cells were harvested the next day and washed three times withPBS, and 10⁷ cells per mouse were injected intravenously for in vivopriming of T cells.

For experiments to monitor cell division, OT-1 splenocytes were labeledwith 5 μM 5- (and 6-) carboxyfluorescein diaceteate succinimidyl ester(CFSE) for 20 minutes at 37° C. prior to adoptive transfer. Followingthree washes with PBS, 10⁷ CFSE-labeled splenocytes were intravenouslyinjected into each mouse. Dendritic cells and T cells were administeredin separate injections. Spleen cells were harvested 2 or 7 days afteradoptive transfer and either analyzed directly by flow cytometry orincubated in culture with various concentrations of ovalbumin for threeadditional days. Cultures were pulsed with ³H-thymidine overnight beforeharvesting and evaluation for ³H incorporation as a measure of T cellactivation.

Competition for binding—PD-1.Ig was acquired from Lieping Chen at theMayo Clinic (Rochester, Minn.). The plasmid encoding PD-1.Ig wasoriginally obtained from Drew Pardoll (Johns Hopkins University,Baltimore, Md.). The plasmid was transformed into CHO cells (ATCC,Manassas, Va.), and PD-1.Ig was isolated from culture supernatants usingprotein G columns (Pharmacia). Dendritic cells were preincubated withPD-1.Ig for 20 minutes at 4° C. before addition of sHIgM12 andsubsequent staining with a fluorescein isothiocyanate- (FITC-)conjugated secondary antibody. For the reverse experiments, cells werepreincubated with sHIgM12 before addition of PD-1.Ig. An isotype controlantibody was used as a control.

Staining of transfected cells—293-T cells and P815 cells were obtainedfrom ATCC. Cells were transiently transfected with expression plasmidsencoding either B7-DC or B7-H1 and stained with sHIgM12, PD-1.Ig, or anisotype control antibody.

Ltk cells (ATCC) were transiently transfected with 2.5 μg of pCDNA3.1(Invitrogen, Carlsbad, Calif.) or 0.5 μg to 10 μg of pCDNA3.1-hB7.DCexpression plasmids. After 48 hours, cells were stained with sHIgM12 orpolyclonal hIgM control. FITC-conjugated anti-hIgM secondary antibodywas added after several washes.

In vivo assays—To evaluate in vivo effects of sHIgM12 on T cellproliferation, mice were treated with 10 μg of sHIgM12 or polyclonalHIgM control on days −1, 0, and +1, and intravenously injected with 1 mgovalbumin on day 0. On day 7, splenocytes were isolated and pulsed withconcentrations of ovalbumin ranging from 1 ng/ml to 1 mg/ml. After threedays of culture in vitro, cells were incubated with 1 μCi of³H-thymidine for 16 hours before harvest and determination of ³Hincorporation.

The effect of sHIgM12 on a lethal tumor cell challenge was evaluated inC57BL/6J mice, C57BL/6-RAG^(−/−) immunodeficient mice, K⁻D⁻ class Iknockout mice, and CD4^(−/−) knockout mice. The animals received 10 μgof sHIgM12, polyclonal HIgM control, or PBS intravenously on days −1, 0,and +1. All mice received a subcutaneous injection of 2×10⁴ B16 melanomacells in the flank on day 0. The presence of tumors was evaluatedstarting on day 10. Data were pooled from three separate trials.Categorical data was analyzed using Chi-square distribution (C57BL/6J)or Fischer exact test (C57BL/6-RAG^(−/−), CD4^(−/−) and K⁻D⁻).

For studies of tumor growth, the width and length of subcutaneous tumorswere measured on day 17 (C57BL/6) or day 13 (C57BL/6-RAG^(−/−)). Theproduct of width and length was used as an estimate of tumor size.Statistical comparisons were made using ANOVA.

To evaluate the persistence of anti-tumor resistance in tumor survivors,C57BL/6 mice that survived otherwise lethal B16 melanoma challenge wererechallenged with 2×10⁴ tumor cells 30 or more days after the primarychallenge. Rechallenge was administered on the opposite flank from theprimary challenge. No further antibody treatments were administered. Asreference points for comparison, naïve animals were treated with sHIgM12or polyclonal HIgM control and challenged with the same dose of tumorcells. Developing tumors were measured (width and length) on day 14, anddata were analyzed by ANOVA.

Cytokine withdrawal assay—Day 5 dendritic cells were plated on 96-wellplates at 2×10⁴ cells per well. Cells were cultured with sHIgM12, A2B5control antibody, or media to a final concentration of 10 μg/ml inRPMI10 with 10 μg/ml granulocyte macrophage-colony stimulating factor(GM-CSF) and 1 ng/ml interleukin-4 (IL-4). Alternatively, dendriticcells were contacted with immobilized PD-1.Ig prior to plating andculturing in RPMI-10 with GM-CSF and IL-4. Cells were cultured for 5days before cytokine withdrawal. For cytokine withdrawal, cells werewashed and cultured in RPMI-10 alone. After 1 hour, Alamar Blue(Biosource International, Camarillo, Calif.) was added to a finalconcentration of 10% (v/v). Readings were taken at 6 hour intervals on aCytoFluor multiplate reader (Series 4000, PerSeptive Biosystems,Framingham, Mass.). The fluorescence plate reader was set to anexcitation wavelength of 520 nm and an emission wavelength of 590 nm.Each data point was done in triplicate.

Assessment of dendritic cell migration to lymph nodes—Bone marrow fromGFP transgenic mice was used to derive GFP⁺ dendritic cells. In someexperiments, the cells were pulsed with aSer-Ile-Ile-Asn-Phe-Glu-Lys-Leu (SEQ ID NO:1) peptide and then treatedwith sHIgM12 or an isotype control antibody for 16 hours or over night.Cells were subcutaneously injected into mice, and then isolated from theipsilateral popliteal and inguinal lymph nodes 48 hours after transfer.Contralateral lymph nodes from both treatment groups served as controls.To measure the number of GFP⁺ dendritic cells that migrated to the lymphnodes, samples were stained with PE-conjugated CD11c antibody andanalyzed by flow cytometry.

To assess the effect of these GFP⁺ dendritic cells, splenocytes fromOT-1 T cell receptor (TcR) transgenic mice (3×10⁵ T cells per well) wereco-cultured for 4 days with titrated numbers of cells from theipsilateral draining lymph nodes of mice treated as described above.Cells were pulsed with 1 mCi of ³H-thymidine per well for the final 16hours of the incubation, and then harvested for measurement of ³Hincorporation. Each group was performed in triplicate.

In related studies, GFP⁺ dendritic cells were mixed with sHIgM12 orcontrol antibody (10 μg/ml) immediately prior to transplantation, andthe migration of the cells to lymph nodes was assessed as describedabove. In another group of studies, the cells were transferred withoutantibody and the mice received three intravenous tail injections of 10μg sHIgM12 or HIgM control on days −1, 0, and +1 relative to thetransplant. Again, lymph nodes were harvested and analyzed as describedabove.

IL-12 measurement—Day 7 bone marrow derived dendritic cells were treatedwith sHIgM12, polyclonal HIgM control, or LPS at a final concentrationof 10 μg/ml. Supernatants were collected 96 hours after stimulation andan ELISA (BD PharMingen, San Diego, Calif.) was performed for the activefraction of IL-12. The supernatant tested for each treatment group waspooled from 6 separate wells. Experimental groups were tested intriplicate and at numerous dilutions, with each demonstrating a similarprofile.

Example 2 A Monoclonal Human IgM Antibody Binds Mouse Dendritic Cells

Dendritic cells were obtained in culture following incubation of mousebone marrow cells in media supplemented with GM-CSF and IL-4. Cells fromseven day cultures were incubated with purified antibodies isolated fromhuman sera, and stained with fluoresceinated goat anti-human antibody aswell as antibodies specific for cell surface molecules typicallyexpressed on dendritic cells. As shown in FIG. 1, the human antibodysHIgM12 bound cells in the cultures that expressed high levels of CD11c,class II, and CD86. Polyclonal human IgM, as well as the other testedmonoclonal antibodies from patients with gammopathies or fromEBV-transformed cell lines did not appreciably bind the dendritic cellpopulations.

To determine when the cell surface determinant recognized by the sHIgM12antibody first appears during the in vitro development of dendriticcells, cultured cells were analyzed by flow cytometry at various timesduring the culture procedure. The determinant first appeared on day 5,approximately 2 days after the appearance of cells expressing highlevels of the dendritic cell marker CD11c. The determinant was expressedat even higher levels in cells cultured in the presence of LPS and CpG,two molecular signals associated with bacterial infection.

Dendritic cells isolated from various tissues were examined to establishwhether endogenous cells express the determinant bound by sHIgM12antibody. Dendritic cells freshly isolated from spleen, thymus, and bonemarrow all were stained by the sHIgM12 antibody. In contrast, most otherbone marrow cells, splenic B cells, splenic T cells, and splenicmacrophages were not appreciably stained by sHIgM12. B cells, T cells,NK cells, and macrophages were activated with LPS or concanavalin A toassess whether activated lymphoid or monocytic cells express theantigen. None of the activated cells from these lineages bound sHIgM12.The sHIgM12 antibody therefore appears to bind a cell surface moleculeexpressed selectively by dendritic cells, and this determinant isexpressed increasingly as the dendritic cells mature and becomeactivated.

Example 3 The sHIgM12 Antibody Potentiates Dendritic Antigen-Presentingfunction

To determine whether binding of sHIgM12 to the surface of dendriticcells influences the pattern of expressed cell surface molecules, day 7dendritic cell cultures were supplemented with 10 μg/ml antibody,incubated overnight, and analyzed by flow cytometry. No changes in cellsurface markers specific to sHIgM12 treatment were observed as comparedto cultures treated with human polyclonal IgM antibodies or othermonoclonal human IgM antibodies.

The antigen-presenting functions of the dendritic cells were assessed invitro. Antibody-treated dendritic cells were pulsed with peptide antigenand used to stimulate naïve antigen-specific T cells freshly isolatedfrom OT-1 and DO-11 transgenic mice. T cell activation was measured byincorporation of ³H-thymidine as described in Example 1. Dendritic cellsthat were pulsed with a class I-binding peptide(Ser-Ile-Ile-Asn-Phe-Glu-Lys-Leu; SEQ ID NO:1) and incubated withpolyclonal HIgM control antibody were able to activate naïve CD8 T cellsfrom OT-1 mice. Dendritic cells treated with the same peptide andincubated with the monoclonal sHIgM12 antibody activated naïve T cellsapproximately 10-fold more effectively, as judged by the number ofantigen-pulsed dendritic cells required to induce the incorporation of³H-thymidine (FIG. 2A). BALB/c dendritic cells pulsed with a peptide(Ile-Ser-Gln-Ala-Val-His-Ala-Ala-His-Ala-Glu-Ile-Asn-Glu; SEQ ID NO:2)presented by class II molecules were even more effective at activatingnaïve T cells freshly isolated from DO-11 TcR transgenic mice. Greaterthan 100-fold more dendritic cells treated with polyclonal HIgM controlantibody were needed to activate T cells to levels observed withsHIgM12-treated dendritic cells (FIG. 2B). These experimentsdemonstrated that the antigen-presenting functions of dendritic cellswere dramatically enhanced by treatment of dendritic cells with sHIgM12.

To assess the requirement for direct contact between dendritic cells andT cells in the potentiation of T cell activation, the two cell types arecultured in compartmentalized tissue culture plates that allow solublefactors to move between chambers but do not allow cellular contactbetween chambers. Alternatively, antibody-depleted supernatants fromdendritic cell cultures treated with sHIgM12 are incubated with culturesof transgenic spleen cells or transgenic spleen cells mixed withdendritic cells pulsed with specific antigen.

The ability of antigen-pulsed, antibody-treated dendritic cells preparedin vitro to stimulate splenic T cells in vivo was evaluated in C3H/H3Jmice. This inbred strain is genetically defective at the TLR-4 locus andconsequently is not responsive to LPS, an activator of dendritic cells.Day 7 cultures of mouse bone marrow-derived dendritic cells wereincubated overnight with chicken ovalbumin and sHIgM12 or polyclonalHIgM control antibody, and 10⁷ cells were intravenously infused intoeach mouse. After seven days, spleen cells were removed from theanimals, incubated in vitro with various amounts of ovalbumin for threedays, and T cell activation was measured by incorporation of³H-thymidine. As shown in FIG. 3, spleen cells from animals that hadreceived sHIgM12-treated dendritic cells responded much more vigorouslyto secondary challenge with ovalbumin than did spleen cells from micethat received dendritic cells treated with polyclonal HIgM control.Dendritic cells treated in culture with sHIgM12 therefore displayedenhanced ability to stimulate T cells in vivo. Because dendritic cellsfrom the TLR-4 deficient mice were responsive to sHIgM12 treatment,possible contamination by LPS was not a factor in these experiments. Inparallel studies, polymixin B was added to the dendritic cell culturesto inactivate potential LPS contaminants. Polymixin B had no influenceon dendritic cell function following treatment with sHIgM12, although itwas effective in reducing maturation of the dendritic cells when LPS wasadded directly to the cultures.

To visualize what was happening to the T cells in vivo, C57BL/6antigen-pulsed, antibody-treated dendritic cells were adoptivelytransferred along with transgenic OT-1 cells into C57BL/6 hosts. OT-1 Tcells were identified in these experiments by probing withK^(b):Ser-Ile-Ile-Asn-Phe-Glu-Lys-Leu (SEQ ID NO:1) tetramers. Spleencells were recovered 2 or 7 days after transfer, and tetramer-positive Tcells were analyzed by flow cytometry to determine their activationstate. T cells stimulated in vivo by dendritic cells pretreated withsHIgM12 expressed substantially higher levels of the activation markersCD44 and CD69 two days after transfer as compared to T cells stimulatedby dendritic cells pretreated with PBS. By day 7, cells remaining in thespleen were less activated, but cells transferred into mice receivingsHIgM12-treated dendritic cells still expressed higher levels of CD44and CD69. Dendritic cells not treated with antigen had no effect on theactivation of transgenic T cells upon adoptive transfer, whetherpretreated with sHIgM12 or not. Treatment with sHIgM12 thereforepotentiated the ability of dendritic cells to activate T cells in vivo.

Example 4 The Pentameric Structure of sHIgM12 Facilitates Potentiationof Dendritic Cells

To test the hypothesis that low affinity IgM antibodies have the abilityto activate cells because they cross-link multiple receptors on thecells surface of targeted cells, monomeric fragments of sHIgM12 wereevaluated for their ability to stain dendritic cells, potentiatedendritic cell function, and block the ability of an intact sHIgM12antibody to potentiate function. IgM monomers were significantly lesseffective than intact sHIgM12 at staining dendritic cells (FIG. 4A).However, the fragments did stain the cells more than polyclonal IgMantibodies, suggesting that they have intact, low affinity bindingsites. Moreover, the antibody fragments were able to block the abilityof intact IgM to potentiate dendritic cell antigen-presenting function(FIG. 4B). Overnight treatment with sHIgM12 monomers did not, however,potentiate the ability of dendritic cells to induce T cells toincorporate ³H-thymidine. The sHIgM12 antibody therefore may function bycross-linking multiple determinants on dendritic cells. The monomericfragments can bind the determinants and thus block the ability of thepentamers to cross-link the relevant cell surface structures.

Example 5 B7-DC is the Cognate Receptor for sHIgM12 on Murine DendriticCells

To determine the identity of the receptor for sHIgM12 on the surface ofdendritic cells, murine bone marrow derived dendritic cells wereincubated with or without a soluble PD-1.Ig fusion protein and stainedwith sHIgM12. Binding of the PD-1 fusion protein attenuated sHIgM12staining to approximately 50% of the level observed in the absence ofPD-1 (FIG. 5A). The reciprocal experiment showed that sHIgM12 alsoreduced the binding of PD-1 to dendritic cells to about 20% of the levelobserved in the absence of sHIgM12 (FIG. 5B). The higher avidity of thepentameric IgM antibody may contribute to the higher degree ofcompetition by sHIgM12.

To investigate whether sHIgM12 can bind to B7-DC, 293T cells weretransfected with a plasmid encoding murine B7-DC. 2×10⁵ cells wereplated and incubated overnight prior to transfection. 2 μg of theexpression plasmid was mixed with 5 μl of FU-GENE (Roche) and incubatedfor 20 minutes in a 37° C. incubator. The mixture was pipetted directlyonto the cells. The cells were cultured for 48 hours at 37° C., and thenstained with either sHIgM12 or a control antibody. Flow cytometryrevealed that approximately 97% of the transfected cells were stained bysHIgM12. Since the PD-1 receptor has been shown to have dual specificityfor B7-DC and B7-H1, P815 cells were transfected with B7-H1 to determinewhether the epitope for sHIgM12 is conserved between the two familymembers. sHIgM12 did not bind to P815 cells expressing B7-H1, indicatingthat the binding to B7-DC is specific.

Example 6 Targeting B7-DC on Dendritic Cells Mediates Potentiation ofImmune Responses to Protein Antigens and Tumors

The systemic effect of sHIgM12 binding to dendritic cells was examinedin vivo. Mice were treated with 10 μg sHIgM12 antibody or polyclonalHIgM control on days −1, 0, and +1, and immunized with 1 mg of ovalbuminon day 0. Seven days after immunization, splenocytes were isolated andassayed for a proliferative response against ovalbumin antigens.Splenocytes treated with polyclonal HIgM control did not mount an immuneresponse against ovalbumin. Treatment with sHIgM12, however, led to highlevels of proliferation in response to titrated amounts of antigen (FIG.6). These data indicate that systemic sHIgM12 has profound immunepotentiating effects, presumably through its interaction with dendriticcells.

The functional role of sHIgM12 cross-linking of B7-DC was examined in ahost immune response against a weakly immunogenic tumor. B 16 melanomais an aggressive, C57BL/6-derived tumor that is known to kill over 95%of immunocompetent mice inoculated subcutaneously with as few as 2×10⁴cells. In this model, palpable tumors typically develop 10 to 12 daysafter inoculation and progress to surface areas in excess of 225 mm² byday 17. Mice were treated with sHIgM12, polyclonal HIgM controlantibody, or PBS injected intravenously on day −1, 0, and +1. All micereceived 2×10⁴ B 16 melanoma cells subcutaneously on day 0. Eleven of 16animals (69%) treated with sHIgM12 remained free from tumors on day 17,whereas only 1 of 13 animals (7%) treated with polyclonal HIgM controlwas tumor free (p<0.001, Table 1). There was no significant differencein tumor incidence in animals receiving polyclonal HIgM control or PBS.Furthermore, tumor growth was significantly inhibited by sHIgM12treatment as compared to treatment with IgM control or PBS (p=0.032,Table 2). The delay in growth was transient, as tumors that developed insHIgM12-treated mice eventually progressed to 225 mm² in size. Somemurine tumor cell lines, particularly those of hematopoietic origin,have been reported to express B7-DC mRNA. The possibility that sHIgM12may act by directly binding to B7-DC on B 16 melanoma cells, however,was ruled out because flow cytometry experiments indicated the antibodydid not appreciably stain tumor cells.

TABLE 1 SHIgM12 treatment protects C57BL/6 mice from lethal challengewith B16 melanoma Strain Treatment Tumor free (day 17) C57BL/6JPolyclonal HIgM control 1/13  PBS 0/13* sHIgM12 11/16**C57BL/6J-RAG^(−/−) Polyclonal HIgM control 0/5  sHIgM12 0/5*  K⁻D⁻Polyclonal HIgM control 0/5  sHIgM12 0/5*  CD4^(−/−) Polyclonal HIgMcontrol 0/9  sHIgM12 0/9*  *no statistical difference; ** p < 0.001

To explore the possibility that host resistance to the lethal tumorchallenge is immune mediated, the same antibody regimen was used inimmunodeficient C57BL/6-RAG^(−/−) mice. Treatment with sHIgM12 had noeffect on the appearance or growth of B16 melanoma in these animals(Tables 1 and 2). The role of CD8 T cells in the host immune response tothe tumors was established using MHC class I knock-out mice (K⁻D⁻).Though these animals have an intact CD4 T cell repertoire, thedeficiency in CD8 T cells abolished the protective effect of sHIgM12.Likewise, the absence of a helper response in CD4 knockout miceabrogated the protective effect of sHIgM12 as all of these micedeveloped palpable tumors akin to mice receiving control treatment. TheB-cell immune response as measured by serum levels of anti-tumorsantibodies was quantitatively indistinguishable between mice receivingsHIgM12 or polyclonal HIgM control. Mice deficient in class II,immunoglobulin, interferon-γ, tumor necrosis factor, perforin, Fas/FasL,and CD40 also are useful for these studies. Such animals are obtainedfrom The Jackson Laboratory.

TABLE 2 sHIgM12 treatment inhibits tumor growth in immunocompetentC57BL/6 mice Avg. Strain Number Treatment tumor size SEM StatisticsC57BL/6 13 Polyclonal 163 mm² ±21.2 HIgM 7 PBS 190 mm² ±22.5 P = 0.666 4sHIgM12  58 mm² ±15.0 P = 0.032 C57BL/6-RAG^(−/−) 5 Polyclonal 150 mm²±22.2 HIgM 5 sHIgM12 194 mm² ±14.8 P = 0.138

The hallmark of an effective adaptive immune response is a vigorousmemory response upon secondary challenge. To determine whether ananamnestic response against B16 tumor antigens was established followingtreatment with sHIgM12, the surviving mice were re-challenged with alethal dose of B16 melanoma cells. As shown in Table 3, mice that hadsurvived for 30 days following the initial tumor challenge displayedsignificant resistance to a secondary challenge (p=0.005). A separategroup of mice was re-challenged after surviving for 90 days after theinitial tumor cell inoculation. In this study, 100% (4/4) mice remainedtumor-free for an additional 30 days following re-challenge. As none ofthe surviving mice received additional treatment with sHIgM12, theresistance to secondary challenge suggests that an effective anti-tumorimmune response was established by treatment with sHIgM12 following theinitial challenge. These data indicate an important role for B7-DC inthe initial priming and subsequent maintenance of a T cell response totumor antigens. In addition to B16 melanoma, weakly antigenic sarcomassuch as Ag104 and MCA102 also are useful for tumor challenge studies ofantibodies such as sHIgM12.

TABLE 3 sHIgM12 treated tumor graft survivors display persistentanti-tumor resistance Strain Number Treatment Avg. tumor size SEMC57BL/6 5 Naïve, polyclonal HIgM  206 mm² ±25.2 5 Naïve, sHIgM12 13.6mm²* ±13.6 5 Tumor survivor, 35.6 mm²* ±20.2 no additional Ab *p < 0.001

Enhancement of dendritic cell vaccine therapy using sHIgM12—SincesHIgM12 significantly enhanced T cell activation at the time of adoptivetransfer into immunocompetent mice (Example 3), it is possible thatsHIgM12 treatment could enhance the protective effect of adoptivelytransferred dendritic cells primed with antigenic tumor-derivedpeptides. To test this possibility, syngeneic dendritic cells pulsedwith the B 16-derived antigenic peptide Trp2₁₈₀₋₁₈₈ are adoptivelytransferred into C57BL/6 mice on the earliest day after tumorinoculation on which antibody treatment alone does not protect the mice.Groups of C57BL/6 mice receive dendritic cells pulsed with either tumorspecific antigenic peptide or an irrelevant peptide along with sHIgM12or polyclonal HIgM control. Alternatively, a B16 melanoma variant thatexpresses chicken ovalbumin is used as an antigen. T cells from OT-1 TcRtransgenic mice are adoptively transferred into mice bearing establishedtumors at various stages (e.g., days +3, +5, +7, +9, and +11) aftertumor challenge. The activation, tumor infiltration, and anti-tumorcytotoxicity of T cells bearing the OT-1 receptor are monitored asanimals are treated with sHIgM12 or polyclonal HIgM control. OT-1 cellsspecific for the surrogate tumor antigen are visualized and isolatedusing T cell-specific class I tetramers such as those generated inresponse to the antigenic Ser-Ile-Ile-Asn-Phe-Glu-Lys-Leu (SEQ ID NO:1)ovalbumin peptide.

Example 7 sHIgM12 Binding to B7-DC Directly Induces Functional Changesin Dendritic Cells

To examine whether binding of sHIgM12 to B7-DC directly affectsdendritic cells biology, dendritic cells were treated in vitro withsHIgM12, polyclonal HIgM control, or LPS. The cells then were analyzedfor their ability to (1) survive in culture following cytokinedeprivation, (2) migrate to draining lymph nodes following adoptivetransfer into naïve animals, and (3) secrete IL-12, a keyimmuno-modulator.

Prolonged survival of dendritic cells could lead to more effectiveinteraction with T cells and thus potentiate immune responses. Thepossibility that sHIgM12 provides a survival signal to dendritic cellsthat would otherwise undergo apoptosis was investigated by cytokinewithdrawal assays. Murine bone marrow-derived dendritic cells wereplated on day 5 into 96-well plates. Cells were cultured with sHIgM12,A2B5 (a control antibody that binds dendritic cells), or media inRPMI-10 containing GM-CSF and IL-4. To achieve cytokines withdrawal,cells were washed in cultured in RPMI-10 alone. Alamar Blue was addedone hour after withdrawal. The metabolism of Alamar Blue was measured at6 hour intervals. The data represent the percentage of cellularmetabolism that was maintained 24 hours after cytokine withdrawal,whereby 100% represents the level of metabolism when dendritic cellswere cultured with GM-CSF and IL-4, and 0% arbitrarily representscomplete cytokine withdrawal.

As shown in FIG. 7A, withdrawal of GM-CSF/IL-4 reduced metabolism to 29%of the level observed in cells cultured in cytokine-supplemented media.Incubating the cells with sHIgM12, however, resulted in 80% maintenanceof metabolism levels 24 hours after cytokine withdrawal. Treatment withA2B5 did not significantly improve metabolism in the cultures ascompared to treatment with media alone (33% vs. 29%). Additionally,treatment with sHIgM12 resulted in larger numbers of viable, Annexin-Vnegative dendritic cells 24 hours after cytokine withdrawal than werefound in comparable cultures treated with control antibodies. Inanalogous experiments, dendritic cells were incubated with a PD-1.Igfusion protein that was immobilized by binding to plastic plates.Treatment with the PD-1.Ig fusion maintained dendritic cell metabolismupon GM-CSF/IL-4 withdrawal in a manner comparable to treatment withsHIgM12 (FIG. 7B), and there was no statistical difference between thetwo groups. Cells contacted with PD-1.Ig maintained metabolism atstatistically higher levels than cells treated with polyclonal HIgMcontrol antibody or PBS (p<0.05). Other IgM antibodies known to bind todendritic cell membranes also are useful for cytokine withdrawalexperiments as described herein. Such antibodies include, for example,the anti-MHC class-I antibodies 28-14-8 (specific for D^(b)) and 28-13-3(specific for K^(b)), as well as the murine oligodendrocyte-bindingantibody 94.03.

The anti-apoptotic activity induced by sHIgM12 indicated that theantibody may induce intracellular signals that inhibit programmed celldeath. To evaluate this possibility, bone marrow cells were culturedwith GM-CSF and IL-4 for 7 days. Cells were treated with sHIgM12,polyclonal HIgM control, or LPS for various lengths of time, stainedwith an antibody against the p65 subunit of NFκB, and subsequentlystained with an antibody against the dendritic cell marker CD11c. Theseexperiments revealed that NFκB was upregulated between 15 and 30 minutesafter addition of sHIgM12. The polyclonal HIgM control had no effect onNFκB levels. The effect of sHIgM12 was specific and was not due to LPScontamination, as dendritic cells from Toll-4 deficient mice did notrespond to LPS treatment but did upregulate NFκB after treatment withsHIgM12. The sHIgM12 antibody therefore may bind to dendritic cells andinduce intracellular signals that block programmed cell death, thusenhancing the ability of the cells to induce T cell responses againstspecific antigens.

To further examine whether binding of sHIgM12 to B7-CD has a directeffect on dendritic cell biology and viability, bone marrow-deriveddendritic cells from mice transgenic for green fluorescent protein (GFP)were treated in vitro prior to adoptive transfer into syngeneic,non-C57BL/6 transgenic mice. Five-fold more GFP⁺, CD11c⁺ dendritic cellswere recovered from draining popliteal and inguinal lymph nodes in micereceiving sHIgM12-treated dendritic cells than those receiving cellstreated with polyclonal HIgM control. The capacity of lymph nodeimmigrants to potentiate an immune response was tested by concomitanttreatment of the dendritic cells with sHIgM12 andSer-Ile-Ile-Asn-Phe-Glu-Lys-Leu (SEQ ID NO:1) peptide prior to adoptivetransfer. In vitro treatment of dendritic cells with anti-B7-DC antibodynot only increased the number of dendritic cells recovered by draininglymph nodes, but also increased by 10-fold the ability of these lymphnode dendritic cells to induce an antigen-specific T cell response.

The ability of sHIgM12 to modulate dendritic cell function in a distanttissue was tested by mixing the antibody with untreated dendritic cellsat the time of transplantation. This experiment was designed todetermine whether dendritic cell migration and survival could beenhanced provided that the antibody reaches the transplantation site.Treatment with the sHIgM12 antibody resulted in enhanced dendritic cellmigration to draining lymph nodes, while the polyclonal HIgM control didnot result in enhanced migration. In related experiments, mice receivedintravenous doses of sHIgM12 or polyclonal HIgM on the day before, theday of, and the day after dendritic cell transplant. Again, themigration of dendritic cells was increased in the mice that receivedsHIgM12, while the control did not have such an effect. Systemicadministration of the antibody therefore is sufficient to influencedendritic cell biology.

IL-12 is a key factor in promoting Th1-type cellular immunity.Production of IL-12 by dendritic cells treated with sHIgM12 was measuredby an ELISA using culture supernatants. Treatment with sHIgM12stimulated nearly 3-fold higher levels of IL-12 p70 release by dendriticcells than did LPS, a strong danger signal. The polyclonal HIgM controldid not elicit detectable levels of IL-12 p70. The increased secretionof IL-12 supports the observation that modulation of B7-DC by sHIgM12strongly potentiates a cellular immune response against even weaklyimmunogenic tumors.

Example 8 sHIgM12 Binds to Human Dendritic Cells

To examine whether sHIgM12 also binds the human B7-DC orthologue, humanmonocyte-derived dendritic cells were stained with sHIgM12 or polyclonalIgM control antibody. sHIgM12 bound weakly to immature dendritic cells.Maturation of the cells with LPS increased the level of sHIgM12 binding,particularly on CD83⁺ cells. Dendritic cells activated with differentstimulation protocols displayed sHIgM12 binding that was increased tovarying degrees: cells activated with LPS were bound by sHIgM12 to ahigh degree, cells activated with TNF-α and IL-1β were bound by sHIgM12to an intermediate degree, and cells activated with IFN-γ were bound bysHIgM12 to a lesser degree.

To determine whether human B7-DC is a ligand for sHIgM12, Ltk fibroblastcells were transiently transfected with a human B7-DC expression plasmidand cultured for 48 hours. sHIgM12 bound to the B7-DC transfected cellsto a significantly higher level than to mock-transfected cells.Furthermore, the level of sHIgM12 binding to L-cells was positivelycorrelated with the amount of B7-DC plasmid used in the transfection.

B7-DC is expressed in a variety of human and murine tumors. To examinewhether sHIgM12 binds to tumor cells, human TP365 glioma cells wereincubated with the antibody. These cells were stained by sHIgM12 at alevel that was significantly higher than the staining by polyclonal IgMcontrol antibody. Furthermore, PCR was used to generate a B7-DC ampliconwith DNA from TP365 cells. The sHIgM12 antibody thus may bind to gliomacells via B7-DC.

Example 9 Production of Recombinant Human IgM Antibodies

Once antibodies of interest such as sHIgM12 are identified, immortalizedsources are generated to sustain these important reagents. A vectorsystem has been developed and used to immortalize a human IgM antibody(sHIgM22) identified in the serum of a Waldenstrom's macroglobulinemiapatient. The amino acid sequence of the antibody was determined from Fvfragments generated from the serum. Since malignant B cells circulate inthe blood of Waldenstrom patients, cDNA encoding the heavy and lightchain genes of the antibody present in highest serum concentrations wassuccessfully isolated. These cDNA sequences were used to generate agenomic human IgM heavy chain gene encoding the variable region derivedfrom the patient antibody and a cDNA-based light chain gene expressedunder control of the cytomegalovirus (CMV) promoter. These antibody genesequences were incorporated into a single vector (FIG. 8) along with aselectable dHfR gene expressed under the control of a SV40 promoter. Thevector bearing the synthetic antibody genes was introduced into F3B6hybridoma cells by electroporation. Methotrexate resistant cells wereselected and amplified by stepping up the amount of methotrexate in theculture medium. A clone expressing 100 μg antibody per ml of supernatantwas recovered. The recombinant antibody displayed all functionalproperties identified for the antibody isolated from the patient serum.

This same procedure is used to generate a recombinant supply of sHIgM12.An amino acid sequence analysis of sHIgM12 has been performed. Since theamino-terminus of the antibody heavy chain was blocked, Fv fragmentswere generated to increase the efficiency of obtaining an amino terminalsequence. The amino terminal sequence of the sHIgM12 heavy chain wasdetermined to be Val-Gln-Leu-Gln-Glu-Ser-Gly-Pro-Gly-Leu-Leu-Lys-Pro-Ser-Glu-Thr-Leu-Arg/Ser-Leu-Thr-Asn (SEQ ID NO:3), while theamino terminal sequence of the light chain was determined to beAsp-Ile-Gln-Met-Thr-Gln-Ser-Pro-Ser-Ser-Leu-Ser-Ala-Ser-Val-Gly-Asp-Arg-Val (SEQ ID NO:4).

CDNA was isolated from the patient's peripheral blood cells, to be usedfor recovering full length cDNA copies of the mRNA encoding sHIgM12. Inorder to ensure than recovered cDNAs truly represent the antibody ofinterest, the amino acid sequence of CDR3 regions of sHIgM12 aredetermined. This is accomplished by proteolytic digestion of the Fvfragments and conventional amino acid sequencing of the digestionproducts. Once the sHIgM12 cDNAs are obtained, they are inserted into avector which is similar to that described above but has been modifiedfor expression of IgM/Kappa antibodies by substituting the light chainconstant region. Recombinant sHIgM12 then is expressed in thehuman/mouse hybridoma line F3B6 as described above. The modified vectorhas been used successfully to express the human antibody rHIgM46.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A purified IgM antibody that binds to B7-DC polypeptides on a cell, wherein said binding results in cross-linking of a plurality of said B7-DC polypeptides, and wherein said IgM. antibody can potentiate an immune response upon administration to a mammal.
 2. The purified IgM antibody of claim 1, wherein said cell is a dendritic cell.
 3. The purified IgM antibody of claim 1, wherein said cell is a tumor cell.
 4. The purified IgM antibody of claim 3, wherein said tumor cell is a glioma tumor cell.
 5. The purified IgM antibody of claim 1, wherein said administration is by injection.
 6. The purified IgM antibody of claim 1, wherein said mammal is a human.
 7. A purified IgM antibody that binds to B7-DC polypeptides on a cell, wherein said binding results in cross-linking of a plurality of said B7-DC polypeptides, and wherein dendritic cells contacted with said molecule exhibit at least one characteristic selected from the group consisting of prolonged longevity, increased NF-κB expression, increased NF-κB translocation to the nucleus, increased ability to activate naive T cells, increased phosphorylation of Akt, increased localization to lymph nodes upon administration to a mammal, maintenance of metabolic rate in culture after cytokine withdrawal, and increased IL-12 secretion.
 8. The purified IgM antibody of claim 7, wherein said activation of naive T cells is measured by detection of one or both of CD44 and CD69 on said T cells, or by incorporation of 3H-thymidine into said T cells.
 9. A composition comprising the purified IgM antibody of claim
 1. 10. The composition of claim 9, further comprising an antigen, wherein said antigen is capable of eliciting an immune response when said composition is administered to a mammal.
 11. The composition of claim 10, wherein said antigen is a tumor antigen.
 12. The composition of claim 10, wherein said antigen is from a pathogen.
 13. The composition of claim 10, wherein said antigen is a component of a killed virus or a component of a killed bacterium.
 14. The composition of claim 10, wherein said antigen is a killed virus or a killed bacterium.
 15. The composition of claim 10, wherein said mammal is a human.
 16. The composition of claim 10, further comprising dendritic cells.
 17. An article of manufacture comprising the IgM antibody of claim
 1. 18. An article of manufacture comprising the composition of claim
 9. 19. The article of manufacture of claim 18, further comprising an antigen, wherein said antigen is capable of eliciting an immune response.
 20. The purified IgM antibody of claim 1, wherein said antibody does not display significant binding to other cell surface polypeptides.
 21. The purified IgM antibody of claim 7, wherein said antibody does not display significant binding to other cell surface polypeptides. 